BIHAREAN BIOLOGIST 6 (1): pp.65-69 ©Biharean Biologist, Oradea, Romania, 2012 Article No.: 121108 http://biozoojournals.3x.ro/bihbiol/index.html

Fumigant toxicity of essential oils of leaves and fruits of Platycladus orientalis to Lasioderma serricorne (F.)

Seyed Mehdi HASHEMI and Seyed Ali SAFAVI*

Department of Plant Protection, Faculty of Agriculture, Urmia University, Urmia, West Azerbaijan, Iran. * Corresponding author, S. Safavi, E-mail: [email protected] Tel +98-441-2972406, Fax: 0441-2779558, P.O. Box 57135-165.

Received: 18. January 2012 / Accepted: 11. April 2012 / Available online: 6. May 2012 / Printed: June 2012

Abstract. Fumigant toxicity of essential oils of leaf and fruit from oriental arborvitae, Platycladus orientalis (Cupressaceae), was investigated against adults of cigarette beetle, Lasioderma serricorne. Fresh leaves and fruits were subjected to hydrodistillation using a Clevenger-type apparatus. The chemical composition of the volatile oils was analyzed by GC-MS. Twenty-six (92.9%) and twenty- three constituents (97.8%) were identified in leaves and fruit oils, respectively. The major components of both leaves and fruit oils were α-Pinene (35.2%, 50.7%), α-Cedrol (14.6%, 6.9%) and Δ-3-Carene (6.3%, 13.8%), respectively. Both oils in the same concentration were tested for their fumigant toxicity on adult insects. Results showed that leaf oil was more toxic than the fruit oil. LC50 values of leaf and fruit oils at 24 h were estimated 43.86 μl/l air and 59.69 μl/l air, respectively. These results suggested that P. orientalis oils may have potential as control agents against L. serricorne.

Key words: Cigarette beetle, fruit oil, fumigant toxicity, Lasioderma serricorne, leaf oil, Platycladus orientalis.

Introduction al. 1948) and also as cough suppressant in traditional Chi- nese medicine (Takao et al. 1985). The present study was The cigarette beetle, Lasioderma serricorne (F.) (Coleoptera: conducted to determine the efficiency of the essential oils Anobiidae), is known to successfully breed and develop on a from P. orientalis as a fumigant in the management of L. serri- variety of grain-based products, spices, and tobacco, and in- corne. fest these commodities during storage, manufacturing and at the retail level (Howe 1957, Bry et al. 1974, Jacob 1992). Con- trol of L. serricorne populations around the world is primar- Materials and methods ily dependent upon continued applications of phosphine Insect culture (PH3) (White & Leesch 1995, Kim et al. 2003). The use of Lasioderma serricorne was reared in glass containers (1 liter) contain- phosphine is increasing due to the convenience of formula- ing wheat flour that was covered by a fine mesh cloth for ventilation. tions, the relatively short-term hazard, and low retention of The culture was maintained in the dark in an incubator set at 27±2°C and 60±5% RH. Parent adults were obtained from laboratory stock residues (Lee et al. 2002). However, PH3 fumigation may be- cultures maintained at the Department of Plant Protection, Urmia come increasingly limited in use because resistance of stored University, Iran. Adult insects, 7-14 days old, were used for fumigant grain insects to phosphine has been discovered in more than toxicity tests. All experiments were carried out under the same envi- 45 countries. (Bell & Wilson 1995, Chaudhry 1995). Naturally ronmental conditions. occurring substances are an alternative to conventional pes- ticides and plant essential oils have traditionally been used Plant material The vegetal organs (leaves and fruits of the P. orientalis) were col- to kill insects (Isman 2000). Essential oils are potential lected during the summer period 2010 from the shrubs cultivated in sources of alternative compounds to currently used fumi- the Department of Horticultural University of Urmia, the region of gants. Essential oils have low toxicity to warm-blooded ani- Nazlo, 12 km in the west of Urmia (latitude: 37° 32', longitude: 45° mals, high volatility and strong toxicity to insect pests (Lee 05'; altitude: 1313 m), Iran. Plant taxonomists in the Department of et al. 2002). Some works have been done to manage L. serri- Biology at Urmia University, confirmed the taxonomic identification corne by using aromatic medicinal plants despite their excel- of plant species. The voucher specimens have been deposited at the lent pharmacological actions (kim et al. 2003, Ebadollahi et herbarium of the Department of Plant Protection at Urmia Univer- sity. al. 2010). Essential oils derived from plant species of Platy- cladus have been evaluated for insecticidal properties (Keita Extraction and analysis of essential oils et al. 2000, Pavela 2005, Jeon et al. 2005). However, no report Fresh leaves and fruits of the plant were separately hydrodistilled in on insecticidal activity of essential oils of P. orientalis against a Clevenger type apparatus where the plant materials were subjected L. serricorne is available. to hydrodistillation. Conditions of extraction were 100 g of fresh sample; 1:10 plant material/water volume ratio, 4 h distillation. An- Platycladus orientalis (L.) Franco [Thuja orientalis L.] lo- hydrous sodium sulphate was used to remove water after extraction. cally named Sarv-e Khomreii or Nosh is an evergreen spe- Extracted oils were transferred to the glass flasks that were filled to cies, which grows naturally in Iran. In addition, this species the top and were kept at the temperature of 4°C in a refrigerator. is widely cultivated as a common ornamental plant in Iran The constituents of P. orientalis essential oils were analyzed by and other countries (Assadi 1998). Platycladus is a genus gas chromatography-mass spectrometry (GC-MS) using a Hewlett- from Family Cupressaceae, with five species, two native to Packard 6890/5972 system with a HP-5MS capillary column (30 m × North America and three native to eastern Asia, they are 0.25 mm; 0.25 μm film thickness). The carrier gas was helium with commonly known as arborvitae (Vines 1987). Platycladus was flow 1 ml/min. The oven temperature was held at 60°C for 3 min, programmed at 6°C /min to 220°C and then held at this temperature an old remedy for delayed menstruation, being also a stimu- for 3 min. Mass spectra were taken at 70 ev. Mass range was from lant to smooth muscles such as those of uterus and bronchial m/z 35-350 amu. The injector temperature was 240°C. Relative per- passages so it is used for treatment of bronchitis (Mabey et centage amounts were calculated from peaks total area by apparatus 66 Hashemi, S.M. & Safavi, S.A. software. The compounds were identified by comparing mass spec- pellent activity in comparison with other oils. Evaluation of tra and retention indices with those in literatures (Adams, 1995) and larvicidal activities of P. orientalis oils against 4th-instar lar- by computer searching followed by matching the mass spectra data vae of Aedes aegypti and Culex pipiens pallens was revealed by with those held in a computer library (Wiley 275.L). Jeon et al. (2005). Larvicidal activities of leaf oils were sig- Bioassay nificantly higher than stem, fruit, and seed oils. The essential To determine the fumigant toxicity of the P. orientalis oils, same con- oils from leaves and fruits at 400 ppm exhibited 100 and centrations of the leaf and the fruit oils including 8, 10.4, 13.5, 17.6 71.6% mortalities against A. aegypti, and 100 and 53.1% and 22.8 μl with 280 ml capacity jars, corresponding to 22.4, 29.1, against C. pipiens, respectively. However, the essential oils of 37.8, 49.2, and 63.8 μl/l air were tested on L. serricorne. They were stems and seeds showed no mortality at 400 ppm. The essen- applied to filter papers (Whatman No. 1, cut into 4 × 5 cm paper strip) and put into 280 ml glass jars. Doses were applied based on tial oils of leaves and fruits exhibited 100 and 18.7% mortali- calculated concentrations. Twenty adults of L. serricorne (7-14 days ties against A. aegypti and 92.1 and 18.7% against C. pipiens at old) were placed in small plastic tubes (3.5 cm diameter and 5 cm 200 ppm, respectively. The observed differences in the ef- height) with open ends covered with cloth mesh. The tubes were fects produced by the essential oils could be due to the pres- hung at the geometrical centre of glass bottles, which were then ence of different secondary metabolites in both vegetal or- sealed with airtight lids. Mortality was determined after 24, 48 and gans (Murray et al. 2005). 96 h from commencement of the exposure. Each concentration and Papachristos & Stamopoulos (2002) tested the essential control were replicated four times. In the control jars, no essential oil of the fruit of P. orientalis against Acanthoscelides obtectus. oils were used. The tests revealed that the oil reduced fecundity, decreased Data analysis egg hatchability, increased neonate larval mortality and ad- Mortality percentages were calculated by the Abbott correction for- versely influences offspring emergence. The oils of P. orien- mula for natural mortality in the untreated control (Abbott, 1925). talis revealed larvicidal activity against two mosquito species Tests were arranged as completely randomized design and data (Jeon et al. 2005). Extracts of P. orientalis have shown insecti- were analyzed by one-way ANOVA using the SAS software version cidal action against larvae of malaria and Japanese encepha- 9.1. Lethal concentrations (LC50 and LC95) and lethal time values (LT50 and LT95) were calculated with SPSS software (version 16.0). litis vector (Sharma et al. 2005). Molluscicidal activity of ex- Comparison of means were done through Tukey’s test (α=0.01). tracts and leaf powder of plant was studied against the snail Lymnaea acuminata (Lamarck) (Singh & Singh 2009). Based on fresh weights, the essential oils of leaves and Results fruits gave yellowish oils with a yield of 0.3% and 0.95%, re-

spectively. GC-MS analyses of the oils identified twenty-six Chemical constituents of essential oils compounds (92.9%) and twenty-three constituents (97.8%) in The main components of both leaves and fruits essential oils the leaf and the fruit oils, respectively (Table 1). Leaf and from P. orientalis were α-Pinene (35.2%, 50.7%), α-Cedrol fruit oils had major compositions similar to those of other P. (14.6%, 6.9%), Δ-3-Carene (6.3%, 13.8%), and Limonene orientalis essential oils analyzed in Iran (Hassanzadeh et al. (6.1%, 1.5%), respectively (Table 1). 2001, Nickavar et al. 2002, Afsharypuor & Nayebzadeh 2009, Bioassay Emami et al. 2011a, b). There are few previous reports on the The insecticidal activity varied with plant-derived material, phytochemical studies of the oil of P. orientalis in other re- different concentrations of the oils and exposure time. The gions of the world (Emami et al. 2011b). Zhu et al. (1995) re- effects of fumigation of essential oils of leaves and fruits of P. ported that the yield of leaf oil of P. orientalis from China orientalis were very toxic on adult of L. serricorne. Probit was 0.3% and had 38 constituents. The major components analysis showed that at exposure time of 24 h, L. serricorne were α-pinene (4.3%), Δ-carene (6.1%) and cedrol (22.3%). was more susceptible to leaf oil with LC50 = 43.86 μl/l air Tanker et al. (1977) showed that the amount and main com- than fruit oil LC50 = 59.69 μl/l air (Tables 2 and 3). Further- ponents of the leaf oil of two different samples of P. orientalis more, with the increase of exposure time to 96 h, mortality collected from Turkey in two different regions of Anatolia increased and LC50 values decreased to 24.84 μl/l air and (Mersin and Ankara) in different times (August and January) 28.6 μl/l air for the leaf and fruits oils, respectively (Tables 2, were different. The main components of the Mersin oil were

3 and Fig. 1). LT50 values and their corresponding informa- a mixture of α-pinene and α-thujene (33.5%), β-Pinene tion were calculated at the different concentrations of oils (16.6%), a mixture of Myrcene and α-phellandrene (12.5%), (Tables 2 and 3). limonene (12.0%), and Δ3-carene (8.7%). The main compo- According to the results of ANOVA, the mortality per- nents of the Ankara January oil sample were a mixture of α- cents significantly increased depending on the increase in pinene and α-thujene (32.0%), β-Pinene (23.1%), sabinene the essential oils concentration. Adults of L. serricorne were (14.3%), limonene (8.2%), and a mixture of myrcene and α- significantly influenced in a different manner being exposed phellandrene (7.11%). While the major constituents of An- to essential oils. kara August sample oil were a mixture of α-pinene and α- thujene (51.8%), limonene (11.3%), β-Pinene (10.8%) and a mixture of myrcene and α-phellandrene (8.5%). The yield Discussion oils were 1.66% for the sample collected from Mersin and 0.79% from Ankara (in January) and 0.84% (in August), re- Essential oil from the leaf oil was more toxic than the fruit oil spectively. on the insect (Fig. 1). Papachristos & Stamopoulos (2002) In comparison with the published data, it can be clearly tested 13 essential oils against Acanthoscelides obtectus (Say). shown that the ingredients of the essential oils of leaves and They reported that the fruit oil of P. orientalis had lowest re- the fruit of P. orientalis are similar, but with differences in

Platycladus orientalis against Lasioderma serricorne 67

Table 1. Main chemical components (%) of the leaf and the fruit oils of Platycladus orientalis.

Compound Retention Index % Leaf oil % Fruit oil α-Pinene 936 35.2 50.7 Sabinene 971 1.5 2.1 Myrcene 993 3.3 3.8 α-Phellandrene 1005 1.6 2.1 Δ3-Carene 1013 6.3 13.8 ρ-Cymene 1021 1.4 2.0 Limonene 1032 6.1 1.5 Terpinolene 1063 2.1 1.7 β-Caryophyllene 1423 5.8 4.1 α-cedrol 1614 14.6 6.9 Total 92.9 97.8

Table 2. Result of the leaf oil probit analysis to calculate LC50, LC95, LT50, and LT95 values.

Insect species Time LC50 LC95 χ2 [df=3] p Intercept Slope 24 55.00 117.13 0.27a 0.96 -3.72 10.01 L. serricorne 48 43.86 102.48 0.36a 0.94 -2.31 9.45 96 24.84 38.73 5.60b 0.13 -6.90 13.53

Concentrations (μl/l air) LT50 [h] LT95 [h] χ2 [df = 1] P Intercept Slope 22.4 104.69 295.10 0.03a 0.86 -2.38 8.65 29.1 89.63 421.66 0.04a 0.83 0.23 7.44 37.8 43.92 109.49 6.27b 0.10 -1.81 9.14 49.2 32.12 105.01 4.51b 0.03 0.19 8.19 63.8 21.19 73.54 1.16a 0.28 0.87 8.04

a Since goodness-of-fit Chi square is not significant (P > 0.15), no heterogeneity factor is used. b Since goodness-of-fit Chi square is significant (P < 0.15), a heterogeneity factor is used.

Table 3. Result of the fruit oil probit analysis to calculate LC50, LC95, LT50, and LT95 values.

Insect species Time LC50 LC95 χ2 [df=3] p Intercept Slope 24 59.69 116.33 0.67a 0.87 -5.08 10.67 L. serricorne 48 50.75 127.29 0.41a 0.93 -2.02 9.11 96 28.56 49.87 0.24a 0.97 -4.89 11.79

Concentrations (μl/l air) LT50 [h] LT95 [h] χ2 [df = 1] P Intercept Slope 22.4 157.97 577.56 0.35a 0.55 -1.42 7.92 29.1 94.16 293.81 0.00a 0.96 -1.57 8.32 37.8 59.72 208.51 1.79a 0.18 -0.38 8.02 49.2 39.13 147.24 3.67b 0.05 0.45 7.85 63.8 24.89 88.55 2.42b 0.12 0.84 7.98

a Since goodness-of-fit Chi square is not significant (P > 0.15), no heterogeneity factor is used. b Since goodness-of-fit Chi square is significant (P < 0.15), a heterogeneity factor is used.

Figure 1. Mean mortality (%) of Lasioderma serricorne exposed to different concentrations of the leaf and the fruit oils of Platycladus orientalis. (Different letters over similar columns indicate significant differences according to Tukey test, P<0.001).

their percentage depending distinctly on the region in which Phellandrene, the absence of Δ-carene, and the high percent- they are grown. Most notable differences observed in the age of α-pinene (Tanker et al. 1977, Zhu et al. 1995). composition of P. orientalis grown in Iran included the low The insecticidal constituents of many plant extracts and es- percentage of α-thujene, β-Pinene, γ-terpinene, and α- sential oils are mainly monoterpenoids (Coats et al. 1991, 68 Hashemi, S.M. & Safavi, S.A.

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